1. Field of the Invention
This invention relates to the analysis of blood sugar metabolism.
2. Information Disclosure Statement
Red blood cells contain hemoglobin, a complex molecule involved in the transport of oxygen and carbon dioxide in the blood. They continuously enter into the circulation from the bone marrow, where they are made. The red blood cells first entering the blood stream are termed reticulocytes, and after the "reticulum" of nucleic acid material is eliminated from the reticulocytes, in the first two days of circulation, the red blood cells continue to circulate, as mature cells, for about 120 days in normal people. After this time the red blood cells are eliminated from circulation and from the body.
The hemoglobin (Hb) molecule consists of heme (a pigment) and globin (a protein). The heme contains iron in the ferrous state. In normal hemoglobin, hemoglobin A (HbA), the protein moiety is formed by two alpha-chain (each 141 amino acids) and two beta chains (each 146 A.A.). Over 100 amino acid sequence variants are known.
It is well known that during their lifetime, red blood cells are bathed in fluid plasma with a continuously changing chemical composition. Some components of the plasma also move freely into and out of the red blood cell through the cell membrane. While in the red blood cell, some of these components exert an influence on the constituent molecules of the red blood cells, particularly on the hemoglobin molecule.
Hemoglobin forms adducts with a variety of molecules, including acetaldehyde, acetate and vitamin B6. Of particular interest in the present instance are the adducts it forms with sugars. These adducts are collectively known as "glycohemoglobins" or "glycated hemoglobins".
The term "glycation" denotes any reaction which links a sugar to a protein. Glycation of a hemoglobin involves the reaction of the carbonyl group of the sugar with a free amine in the hemoglobin. Usually, this free amine is the amino terminal of the beta chain, but it may be the amino terminal of the alpha chain, or a side chain amino group of a lysine residue. The resulting moiety is known as an aldimine. Because there is a hydroxyl group on the carbon adjacent to the carbonyl carbon, a second reaction, the Amadori rearrangement, is possible, which shifts the double bond from C-1 to C-2, forming a ketoamine.
Sugars which form natural adducts with hemoglobin include glucose, 5-deoxy-xylose-1-phosphate, galactose and glucose-6-phosphate. The corresponding keto sugar amines are fructosamine, 5-deoxy-xylulose-1-phosphate, galactulose and fructose-6-phosphate.
When human hemolysate is chromatographed on ion exchange resins, certain negatively charged minor components are eluted before the main HbA peak. In order of elution, these are A1a1, A1a2, A1b, A1c and A1d, and are collectively known as the A1 fraction. These A1 fractions include glycated hemoglobins and possibly other hemoglobin adducts and variants. The A1c fraction includes the glucose and fructosamine adducts of hemoglobin.
Higgins and Bunn (1981) describe the formation of A1c as "nearly irreversible", following their group's earlier report that the Amadori rearrangement is "slightly reversible" (Bunn, et al., 1976). Based on these reports, a person of ordinary skill would disregard "reversibility" as a complicating factor. While, in 1985, Mortenson suggested taking reversibility into account, this is not the current practice. Moreover, Mortenson teaches that there are no age-related differences in HbA1c content among erythrocytes.
The rate of glycation is dependent on blood sugar concentrations. For this reason, glycated hemoglobin measurements have been used to provide an estimate of mean glycemic control over the four to twelve weeks preceding the test. The level of glycated hemoglobin in a red blood cell increases with the age of the red blood cell (and hence the duration of its exposure to blood sugar) and with the mean plasma glucose concentration over the circulating life span of the cell. Traditional glycated hemoglobin measurements cannot detect acute alterations in glycemic status, since such fluctuations will be averaged out as a result of the spread of red blood cell ages in the sample.
Saunders, U.S. Pat. No. 4,835,097 (1989) suggests that the day-to-day control of blood sugar by a diabetic can be determined from a single blood sample by correlating glycohemoglobin levels with blood cell ages on a cell-by-cell basis. While Saunders recognizes that if the reaction is slowly reversible, the history preserved by the oldest blood cells will be lost, he does not describe any means of correcting the data to minimize the distortion of the data by such reversibility.